PROJECT SUMMARY Epigenetic regulation has been shown to play pivotal roles in neurodevelopmental and neuropsychiatric disorders. In addition to DNA and histone modifications, more than 150 post-transcriptionally modified ribonucleosides have been identified in various types of RNA. Furthermore, recent studies have suggested that post-transcriptional messenger RNA (mRNA) modifications are dynamically regulated and significantly impact the outcomes of gene expression. These dynamic RNA modifications represent a critical new realm for gene expression regulation in the form of ?RNA epigenetics? or ?Epitranscriptomics?. Among different RNA modifications, our published and unpublished works suggest that m6A, m3C and m1A are dynamic and could play important roles during neurodevelopment and, potentially, contribute to developmental pathology if dysregulated. Autism spectrum disorder (ASD) is a clinically heterogeneous group of developmental disorders frequently characterized by impaired social relationships, impaired language and communication, a limited range of interests and stereotypic behaviors. We have found that ASD-linked genes are subject to extensive RNA modifications during human brain development. We have further developed robust technologies and pipelines to ?quantitatively? profile/map these RNA specific modifications at the transcriptome-wide level in humans. In this proposed PsychENCODE study, we will use postmortem tissue and human induced pluripotent stem cell-derived organoids to systematically map three distinct mRNA/lncRNA modifications (m6A, m3C and m1A) during normal human brain development and identify any differential patterns between unaffected donors and patients with ASD. These datasets will provide a blueprint of how RNA marks impact the global transcriptome and we will annotate prominent modifications on key transcripts important for brain development and function. Our proposed work will identify and functionally annotate epitranscriptome marks critical to post- transcriptional gene expression regulation across human brain development and adulthood. A systematic analysis of these RNA modifications in the context of human brain development and ASD could provide new functional insights into critical nodes of vulnerability for dysregulated neural development.